Nucleic acid labeling compounds

ABSTRACT

Nucleic acid labeling compounds are disclosed. The compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional patentApplication Serial No. 60/275,202, filed on Mar. 12, 2001, which isherein incorporated by reference for all purposes.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] This invention was made with Government support under contract70NANB5H1031 awarded by the Advanced Technology Program of the NationalInstitute of Standards and Technology. The Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

[0003] Gene expression in diseased and healthy individuals is oftentimesdifferent and characterizable. The ability to monitor gene expression insuch cases provides medical professionals with a powerful diagnostictool. This form of diagnosis is especially important in the area ofoncology, where it is thought that the overexpression of an oncogene, orthe underexpression of a tumor suppressor gene, results intumorogenesis. See Mikkelson et al. J. Cell. Biochem. 1991, 46, 3-8.

[0004] One can indirectly monitor gene expression, for example, bymeasuring a nucleic acid (e.g., mRNA) that is the transcription productof a targeted gene. The nucleic acid is chemically or biochemicallylabeled with a detectable moiety and allowed to hybridize with alocalized nucleic acid probe of known sequence. The detection of alabeled nucleic acid at the probe position indicates that the targetedgene has been expressed. See, e.g., International ApplicationPublication Nos. WO 97/27317, WO 92/10588 and WO 97/10365.

[0005] The labeling of a nucleic acid is typically performed bycovalently attaching a detectable group (label) to either an internal orterminal position. Scientists have reported a number of detectablenucleotide analogues that have been enzymatically incorporated into anoligo- or polynucleotide. Langer et al., for example, disclosedanalogues of dUTP and UTP that contain a covalently bound biotin moiety.Proc. Natl. Acad. Sci. USA 1981, 78, 6633-6637. The analogues, shownbelow, possess an allylamine linker arm that is attached to the C-5position of the pyrimidine ring. The dUTP and UTP analogues, wherein Ris H or OH, were incorporated into a polynucleotide.

[0006] Petrie et al. disclosed a dATP analogue,3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5′-triphosphate.Bioconjugate Chem. 1991, 2, 441-446. The analogue, shown below, ismodified at the 3-position with a linker arm that is attached to abiotin moiety. Petrie et al. reported that the compound wherein R isbiotin is incorporated into DNA by nick translation.

[0007] Prober et al. disclosed a set of four dideoxynucleotides, eachcontaining a succinylfluorescein dye. Science 1987, 238, 336-341. Thedideoxynucleotides, one of which is shown below, were enzymaticallyincorporated into an oligonucleotide through a template directedextension of a primer. The compounds provided for a DNA sequencingmethod based on gel migration.

[0008] Herrlein et al. disclosed modified nucleoside trisphosphates ofthe four DNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds,one of which is shown below, contain a 3′-amino group containingradioactive or fluorescent moieties. Herrlein et al. further describedthe use of the nucleoside analogues as DNA chain terminators.

[0009] Cech et al. disclosed 3′-amino-functionalized nucleosidetriphosphates. Collect. Czech. Chem. Commun. 1996, 61, S297-S300. Thecompounds, one of which is shown below, contain a fluorescein attachedto the 3′-position through an amino linker. Cech et al. proposed thatthe described functionalized nucleosides would be useful as terminatorsfor DNA sequencing.

[0010] The development of novel nucleic acid labeling compounds that areeffectively incorporated into a nucleic acid to provide a readilydetectable composition would benefit genetic analysis technologies. Itwould aid, for example, in the monitoring of gene expression and thedetection and screening of mutations and polymorphisms. Such a compoundshould be suitable for enzymatic incorporation into a nucleic acid.Furthermore, the nucleic acid to which the labeling compound is attachedshould maintain its ability to bind to a probe, such as a complementarynucleic acid.

[0011] Although nucleic acid labeling compounds for use as couplingagents for probes are available there is a continuing need foradditional compounds that are more efficient labeling compounds. Therealso exists a need for compounds that have increased solubility. Thiswill make the compounds more useful for monitoring gene expression.

SUMMARY OF THE INVENTION

[0012] The present invention relates to nucleic acid labeling compounds.More specifically, the invention provides compounds containing adetectable moiety. The invention also provides methods of making thesecompounds. It further provides methods of attaching the compounds to anucleic acid. The nucleic acid labeling compounds or the presentinvention are effectively incorporated into a nucleic acids to providereadily detectable compositions that are useful for genetic analysistechnologies. These compounds and the detectable compositions can aid,for example, in the monitoring of gene expression and the detection andscreening of mutations and polymorphisms. Thus, the compounds of theinvention are suitable for enzymatic incorporation into nucleic acids.Furthermore, the nucleic acids to which the labeling compound areattached maintain their ability to bind to a probe, such as, for examplea complementary nucleic acid.

[0013] The present invention provides nucleic acid labeling compoundsthat are capable of being enzymatically incorporated into a nucleicacid. The nucleic acids to which the compounds are attached maintaintheir ability to bind to a complementary nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 illustrates a non-limiting set of template moieties.

[0015] FIGS. 2-10 illustrate synthetic routes to fluorescein and biotinlabeled compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The nucleic acid labeling compounds of the present invention havethe following structure:

A—T—H_(c)—L—(M)_(m)—Q, or A—T(H_(c))—L—(M)_(m)—Q, or A—T—Q

[0017] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; T isa template moiety; H_(c) is a heterocyclic group; L is a linker moiety;M is a connecting group; and Q is a detectable moiety.

[0018] In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

[0019] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; X isO, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, hydrogen, alkylor aryl; Y is hydrogen, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is hydrogen,alkyl or aryl; Z is hydrogen, N₃, F, OR₉, SR₉ or NHR₁₀, wherein R₁₀ ishydrogen, alkyl or aryl; L is a linker moiety; Q is a detectable moiety;and M is a connecting group, wherein m is an integer ranging from 0 toabout 20.

[0020] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉,wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, F or OR₁₀,wherein R₁₀ is hydrogen, alkyl or aryl; and L is amido alkyl and M is—(M₁)_(a)—(M₂)_(b)— wherein a and b are independently integers from 0 toabout 5 and the sum of a and b is not greater than 15.

[0021] In another embodiment, L is —(CH₂)_(n)C(O)NR₃—, wherein R₃ ishydrogen, alkyl or aryl and n is an integer ranging from about 1 toabout 10; M₁ is —(CH₂)_(i)O— and M₂ is —(CH₂)_(j)NH—, wherein i and jare independently integers from 1 to about 5.

[0022] In another embodiment, Y is H or OH; Z is H or OH; —L is—CH₂C(O)NH—; M, is —(CH₂CH₂O)₃— and M₂ is —CH₂CH₂NH—; and Q is biotin ora carboxyfluorescein.

[0023] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—(CH₂CH₂O)₃—CH₂CH₂NH—; and Q is biotin or Y is OH; Z is OH;—L—(M)_(m)— is —CH₂C(O)NH—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is carboxyfluorescein.

[0024] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉,wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, F or OR₁₀,wherein R₁₀ is hydrogen, alkyl or aryl; and L is amido alkyl and M is—(M₁)_(a)—(M₂)_(b)—(M₃)_(c)—(M4)_(d)— wherein a, b, c, and d areindependently integers from 0 to about 5 and the sum of a, b, c, and dis not greater than 15.

[0025] In another embodiment, L is —(CH₂)_(n)C(O)NR₃—, wherein R₃ ishydrogen, alkyl or aryl and n is an integer ranging from about 1 toabout 10; each M is independently —C(O)(CH₂)_(k)O—, —(CH₂)_(i)O— or—(CH₂)_(J)NH—, wherein i, j and k are independently integers from 1 toabout 5.

[0026] In another embodiment, L is —CH₂C(O)NR₃—, wherein R₃ is hydrogenor, alkyl; M₁ is —(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃ is —(CH₂CH₂O)₃— andM₄ is —(CH₂)₂NH—.

[0027] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—CH₂CH₂NH—C(O)CH₂CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin.

[0028] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—CH₂CH₂NH—; m is 1; and Q is carboxyfluorescein.

[0029] In another embodiment, L is —(CH₂)₂C(O)NR₃—, wherein R₃ ishydrogen or, alkyl; M₁ is —(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃ is—(CH₂CH₂O)₃— and M₄ is —(CH₂)₂NH—.

[0030] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—CH₂CH₂NH—C(O)CH₂CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin.

[0031] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂CH₂C(O)NH—CH₂CH₂NH—; m is 1; and Q is carboxyfluorescein.

[0032] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉,wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, F or OR₁₀,wherein R₁₀ is hydrogen, alkyl or aryl; and L is functionalized alkyland M is —(M₁)_(a)—(M₂)_(b)—(M₃)_(c)−(M₂)_(d)— wherein a, b, c, and dare independently integers from 0 to about 5 and the sum of a, b, c, andd is not greater than 15.

[0033] In another embodiment, L is —(CH₂)_(n)O—, wherein n is an integerranging from about 1 to about 10; each M is independently—C(O)(CH₂)_(k)O—, —(CH₂)_(i)O— or —(CH₂)_(j)NH—, wherein i, j and k areindependently integers from 1 to about 5.

[0034] In another embodiment, L is —CH₂O—; M₁ is —(CH₂)₂NH—, M₂ is—C(O)(CH₂)₂O—, M₃ is —(CH₂CH₂O)₃— and M₄ is —(CH₂)₂NH—.

[0035] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH₂O—CH₂CH₂NH—C(O)(CH₂)₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin or Y is OH;Z is OH; —L—(M)_(m)— is —CH₂O—CH₂CH₂NH—; m is 1; and Q iscarboxyfluorescein.

[0036] In another embodiment, L is —(CH₂)_(n)NR₃—, wherein R₃ ishydrogen, alkyl or aryl and n is an integer ranging from about 1 toabout 10; each M is independently —C(O)(CH₂)_(k)O—, —(CH₂)_(i)O— or—(CH₂)_(j)NH—, wherein i, j and k are independently integers from 1 toabout 5.

[0037] In another embodiment, L is —(CH₂)₆NH—; M₁ is —(CH₂)₂NH—, M₂ is—C(O)(CH₂)₂O—, and M₃ is —(CH₂CH₂O)₃—. In another embodiment, Y is OH; Zis OH; —L—(M)_(m)— is —(CH₂)₆NH—C(O)(CH₂)₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; and Qis biotin or Y is OH; Z is OH; L is —(CH₂)₆NH—; m is 0; and Q iscarboxyfluorescein.

[0038] In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉,wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, F or OR₁₀,wherein R₁₀ is hydrogen, alkyl or aryl; and L is alkenyl alkyl and M is—(M₁)_(a)—(M₂)_(b)—(M₃)_(c)—(M₄)_(d)— wherein a, b, c, and d areindependently integers from 0 to about 5 and the sum of a, b, c, and dis not greater than 15.

[0039] In another embodiment, L is—structure —CH═CH—(CH₂)_(n)C(O)N(R₆)—,wherein R₆ is hydrogen, alkyl or aryl and n is an integer ranging fromabout 0 to about 10; each M is independently —C(O)(CH₂)_(k)O—,—(CH₂)_(i)O— or —(CH₂)_(j)NH—, wherein i, j and k are independentlyintegers from 1 to about 5.

[0040] In another embodiment, L is —CH═CH—C(O)N(H); M₁ is —(CH₂)₂NH—, M₂is —C(O)(CH₂)₂O—, M₃ is —(CH₂CH₂O)₃— and M₄ is —(CH₂)₂NH—.

[0041] In another embodiment, Y is OH; Z is OH; —L—(M)_(m)— is—CH═CH—C(O)NH—CH₂CH₂NH—C(O)(CH₂)₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin orY is OH; Z is OH; —L—(M)_(m)— is —CH═CH—C(O)NH—CH₂CH₂NH—; m is 1; and Qis carboxyfluorescein.

[0042] In another embodiment, the nucleic acid labeling compounds havethe following structure:

[0043] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; L isa linker moiety; Q is a detectable moiety; and M is a connecting group,wherein m is an integer ranging from 0 to about 3.

[0044] In another embodiment, A is H or H₄O₉P₃—;, L is alkanoyl; and Qis biotin or a carboxyfluorescein; M is —NR₃— where R₃ is hydrogen oralkyl, and wherein m is 1 or 0.

[0045] In another embodiment, L is —C(O)(CH₂)_(n)— wherein n is aninteger ranging from about 1 to about 10; M is —NH—; Q is biotin or acarboxyfluorescein.

[0046] In another embodiment, —L—(M)_(m)— is —C(O)(CH₂)₆—NH—; and Q isbiotin or —L—(M)_(m)— is —C(O)(CH₂)₆—NH—; and Q is carboxyfluorescein.

[0047] In another embodiment, the nucleic acid labeling compounds havethe following structures:

[0048] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; X₁is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, hydrogen,alkyl or aryl; R₁₅ is hydrogen, alkyl or aryl; Y, is hydrogen, N₃, F,OR₉, SR₉ or NHR₉, wherein R₉ is hydrogen, alkyl or aryl; Z₁ is hydrogen,N₃, F, OR₉, SR₉ or NHR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl;wherein one of Y, or Z, is a group having the formula —X₂L—(M)_(m)—Q; X₂is O, S, or NR₁₆, and R₁₆ is hydrogen, alkyl or aryl; L is a linkermoiety; Q is a detectable moiety; and M is a connecting group, wherein mis an integer ranging from 0 to about 20.

[0049] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; Y₁ ishydrogen, OR₉, wherein R₉ is hydrogen, or alkyl, or —NR₁₆—L—(M)_(m)—Q;wherein R₁₆ is hydrogen, alkyl or aryl; Z₁ is hydrogen OR₁₀, wherein R₁₀is hydrogen, or alkyl, or —NR₁₆—L—(M)_(m)—Q, wherein R₁₆ is hydrogen, oralkyl; L is N-alkyl amido; R₁₅ is hydrogen or alkyl; M is —(CH₂)_(n)NR₃—where R₃ is hydrogen or alkyl, and m is from 1 to about 15.

[0050] In another embodiment, Y₁ is hydrogen or OH; Z₁ is—NH—L—(M)_(m)—Q; L is —(CH₂)_(n)NHC(O)— where n is an integer from 1 toabout 10; M is —NH(CH₂)_(p)— where p is an integer from 1 to about 10;R₁₅ is hydrogen or methyl; and Q is biotin or a carboxyfluorescein.

[0051] In another embodiment, Y₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.

[0052] In another embodiment, Y₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is a carboxyfluorescein.

[0053] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.

[0054] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is a carboxyfluorescein.

[0055] In another embodiment, Y₁ is —NH—L—(M)_(m)—Q; Z, is hydrogen orOH; L is —(CH₂)_(n)NHC(O)— where n is an integer from 1 to about 10; Mis —NH(CH₂)_(p)— where n is an integer from 1 to about 10; R₁₅ ishydrogen or methyl; and Q is biotin or a carboxyfluorescein.

[0056] In another embodiment, Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.

[0057] In another embodiment, of the nucleic acid labeling compound, Z₁is OH; R₁₅ is hydrogen; —L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q isa carboxyfluorescein.

[0058] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(n)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.

[0059] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is a carboxyfluorescein.

[0060] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; Y₁ ishydrogen, OR₉, wherein R₉ is hydrogen, or alkyl, or —O—L—(M)_(m)—Q; Z,is hydrogen OR₁₀, wherein R₁₀ is hydrogen, or alkyl, or —O—L—(M)_(m)—Q,or alkyl; L is alkylene; and m is from 1 to about 10.

[0061] In another embodiment, Y₁ is hydrogen or OH; Z, is—O—L—(M)_(m)—Q; L is —(CH₂)_(n)— where n is an integer from 1 to about12; M is —NH—; R₁₅ is hydrogen or methyl; and Q is biotin or acarboxyfluorescein.

[0062] In another embodiment, Y₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is biotin.

[0063] In another embodiment, wherein Y₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —(CH₂)₆—NH—; and Q is a carboxyfluorescein.

[0064] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is biotin.

[0065] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is a carboxyfluorescein.

[0066] In another embodiment, Y₁ is —O—L—(M)_(m)—Q; Z, is hydrogen orOH; L is —(CH₂)_(n)— where n is an integer from 1 to about 12; M is—NH—; R₁₅ is hydrogen or methyl; and Q is biotin or acarboxyfluorescein.

[0067] In another embodiment, Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is biotin.

[0068] In another embodiment, Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is a carboxyfluorescein.

[0069] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is biotin.

[0070] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is a carboxyfluorescein.

[0071] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; Y₁ ishydrogen, OR₉, wherein R₉ is hydrogen, or alkyl, or —S—L—(M)_(m)—Q; Z₁is hydrogen OR₁₀, wherein R₁₀ is hydrogen, or alkyl, or —S—L—(M)_(m)—Q;L is alkylene; X₂ is S; and in is from 1 to about 10.

[0072] In another embodiment, Y₁ is hydrogen or OH; Z₁ is—S—L—(M)_(m)—Q; L is —S—(CH₂)_(n)— where n is an integer from 1 to about10; M is —NH—; R₁₅ is hydrogen or methyl; and Q is biotin or acarboxyfluorescein.

[0073] In another embodiment, Y₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is biotin.

[0074] In another embodiment, Y₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.

[0075] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is biotin.

[0076] In another embodiment, Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.

[0077] In another embodiment, Y₁ is —S—L—(M)_(m)—Q; Z, is hydrogen orOH; L is —S—(CH₂)_(n)— where n is an integer from 1 to about 10; M is—NH—; R₁₅ is hydrogen or methyl; and Q is biotin or acarboxyfluorescein.

[0078] In another embodiment, —Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is biotin.

[0079] In another embodiment, Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.

[0080] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is biotin.

[0081] In another embodiment, Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.

[0082] In another embodiment, the nucleic acid labeling compounds havethe following structure:

[0083] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; X₁is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, hydrogen,alkyl or aryl; X₂ is a bond or alkylene; Q is a detectable moiety; and Gis —L—(M)_(m)— where L is a linker moiety and each M a connecting group,where m is from 0 to about 20.

[0084] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; X₂ is a bond;and G is —C(O)NR₃—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NR₃—, where R₃ ishydrogen or alkyl, and m and n are independently an integer from 1 toabout 15.

[0085] In another embodiment, G is

—C(O)NH—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NH—

[0086] where m is from 1 to about 6 and n is from 1 to about 4.Preferably, n is 3 or 4and m is 5 or 6.

[0087] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; X₂ is CH₂; andG is —C(O)NR₃—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NR₃—, where R₃ ishydrogen or alkyl, and m and n are independently an integer from 1 toabout 15.

[0088] In another embodiment, A is H or H₄O₉P₃—; X₁ is O; X₂ is CH₂; andG is —C(O)NR₃—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NR₃—, where R₃ ishydrogen or alkyl, and m and n are independently an integer from 1 toabout 15.

[0089] The present invention also provides nucleic acid derivativesproduced by coupling a nucleic acid labeling compound with a nucleicacid and hybridization products comprising the nucleic acid derivativesbound to a complementary probe.

[0090] The present invention also provides nucleic acid derivativesproduced by coupling one of the nucleic acid labeling compounds of theinvention with a nucleic acid and the hybridization products comprisingthe nucleic acid derivatives bound to a complementary probe. Thehybridization product formed from the nucleic acid derivatives comprisethe nucleic acid derivative bound to a complementary probe. In oneembodiment, the probe is attached to a glass chip.

[0091] The present invention also provides methods of synthesizingnucleic acid derivatives by attaching a nucleic acid labeling compoundto a nucleic acid. It further provides methods of detecting nucleicacids involving incubating the nucleic acid derivatives with a probe.

[0092] In yet another embodiment, the methods involve the steps of: (a)providing at least one nucleic acid coupled to a support; (b) providinga labeled moiety capable of being coupled with a terminal transferase tosaid nucleic acid; (c) providing said terminal transferase; and (d)coupling said labeled moiety to said nucleic acid using said terminaltransferase.

[0093] In still another embodiment, the methods involve the steps of:(a) providing at least two nucleic acids coupled to a support; (b)increasing the number of monomer units of said nucleic acids to form acommon nucleic acid tail on said at least two nucleic acids; (c)providing a labeled moiety capable of recognizing said common nucleicacid tails; and (d) contacting said common nucleic acid tails and saidlabeled moiety.

[0094] In still yet another embodiment, the methods involve the stepsof: (a) providing at least one nucleic acid coupled to a support; (b)providing a labeled moiety capable of being coupled with a ligase tosaid nucleic acid; (c) providing said ligase; and (d) coupling saidlabeled moiety to said nucleic acid using said ligase.

[0095] This invention also provides compounds of the formulas describedherein.

[0096] Definitions

[0097] The following definitions are used, unless otherwise described.Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl,alkylaryl, etc. denote both straight and branched alkyl groups; butreference to an individual radical such as “propyl” embraces only thestraight chain radical, a branched chain isomer such as “isopropyl”being specifically referred to. Aryl includes a phenyl radical or anortho-fused bicyclic carbocyclic radical having about nine to ten ringatoms in which at least one ring is aromatic. Heteroaryl encompasses aradical attached via a ring carbon of a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is H, O, (C₁-C₄)alkyl,phenyl or benzyl, as well as a radical of an ortho-fused bicyclicheterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene diradical thereto.

[0098] “Alkyl” refers to a straight chain, branched or cyclic chemicalgroups containing only carbon and hydrogen. Alkyl groups include,without limitation, ethyl, propyl, butyl, pentyl, cyclopentyl and2-methylbutyl. Alkyl groups are unsubstituted or substituted with 1 ormore substituents (e.g., halogen, alkoxy, amino).

[0099] “Alkylene” refers to a straight chain, branched or cyclicchemical group containing only carbon and hydrogen. Alkyl groupsinclude, without limitation, ethylene, propylene, butylene, pentylene,and 2-methylbutylene. Alkyl groups are unsubstituted or substituted with1 or more substituents (e.g., halogen, alkoxy, amino).

[0100] “Aryl” refers to a monovalent, unsaturated aromatic carbocyclicgroup. Aryl groups include, without limitation, phenyl, naphthyl,anthryl and biphenyl. Aryl groups are unsubstituted or substituted with1 or more substituents (e.g. halogen, alkoxy, amino). “Arylene” refersto a divalent aryl group.

[0101] “Amido” refers to a chemical group having the structure—C(O)NR₃—, wherein R₃ is hydrogen, alkyl or aryl. Preferably, the amidogroup is of the structure —C(O)NR₃— where R3 is hydrogen or alkyl havingfrom about 1 to about 6 carbon atoms. More preferably, the amido alkylgroup is of the structure —C(O)NH—.

[0102] “Alkanoyl” refers to a chemical group having the structure—(CH₂)_(n)C(O)—, n is an integer ranging from 0 to about 10. Preferably,the alkanoyl group is of the structure —(CH₂)_(n)C(O)—, wherein n is aninteger ranging from about 2 to about 10. More preferably, the alkanoylgroup is of the structure —(CH₂)_(n)C(O)—, wherein n is an integerranging from about 2 to about 6. Most preferably, the alkanoyl group isof the structure —CH₂C(O)—.

[0103] “Alkyl amido” refers to a chemical group having the structure—R₄C(O)NR₃—, wherein R₃ is hydrogen, alkyl or aryl, and R₄ is alkyleneor arylene. Preferably, the alkyl amido group is of the structure—(CH₂)_(n)C(O)NH—, wherein n is an integer ranging from about 1 to about10. More preferably, n is an integer ranging from about 1 to about 6.Most preferably, the alkyl amido group has the structure —(CH₂)₂C(O)NH—or the structure —CH₂C(O)NH—.

[0104] “N-Amido alkyl” refers to a chemical group having the structure—C(O)NR₃R₄—, wherein R₃ is hydrogen, alkyl or aryl, and R₄ is alkyleneor arylene. Preferably, the N-amido alkyl group is of the structure—C(O)NH(CH₂)_(n)R₅—, wherein n is an integer ranging from about 2 toabout 10, and R₅ is O, NR₆, or C(O), and wherein R₆ is hydrogen, alkylor aryl. More preferably, the N-amido alkyl group is of the structure—C(O)NH(CH₂)_(n)N(H)—, wherein n is an integer ranging from about 2 toabout 6. Most preferably, the N-amido alkyl group is of the structure—C(O)NH(CH₂)₄N(H)—.

[0105] “Alkynyl alkyl” refers to a chemical group having the structure—C≡C—R₄-, wherein R₄ is alkyl or aryl. Preferably, the alkynyl alkylgroup is of the structure —C≡C—(CH₂)_(n)R₅—, wherein n is an integerranging from 1 to about 10, and R₅ is O, NR₆ or C(O), wherein R₆ ishydrogen, alkyl or aryl. More preferably, the alkynyl alkyl group is ofthe structure —C≡C—(CH₂)_(n)N(H)—, wherein n is an integer ranging from1 to about 4. Most preferably, the alkynyl alkyl group is of thestructure —C≡C—CH₂N(H)—.

[0106] “Alkenyl alkyl” refers to a chemical group having the structure—CH═CH—R₄—, wherein R₄ is a bond, alkyl or aryl. Preferably, the alkenylalkyl group is of the structure —CH═CH—(CH₂)_(n)R₅—, wherein n is aninteger ranging from 0 to about 10, and R₅ is O, NR₆, C(O) or C(O)NR₆,wherein R₆ is hydrogen, alkyl or aryl. More preferably, the alkenylalkyl group is of the structure —CH═CH—(CH₂)_(n)C(O)NR6—, wherein n isan integer ranging from 0 to about 4. Most preferably, the alkenyl alkylgroup is of the structure —CH═CH—C(O)N(H)—.

[0107] “Functionalized alkyl” refers to a chemical group of thestructure —(CH₂)_(n)R₇—, wherein n is an integer ranging from 1 to about10, and R₇ is O, S, NH or C(O). Preferably, the functionalized alkylgroup is of the structure —(CH₂)_(n)C(O)—, wherein n is an integerranging from 1 to about 4. More preferably, the functionalized alkylgroup is of the structure —CH₂C(O)—.

[0108] “Alkoxy” refers to a chemical group of the structure—O(CH₂)_(n)R₈—, wherein n is an integer ranging from 2 to about 10, andR₈ is a bond, O, S, NH or C(O). Preferably, the alkoxy group is of thestructure —O(CH₂)_(n)—, wherein n is an integer ranging from 2 to about4. More preferably, the alkoxy group is of the structure —OCH₂CH₂—.

[0109] “Alkyl thio” refers to a chemical group of the structure—S(CH₂)_(n)R₈—, wherein n is an integer ranging from 1 to about 10, andR₈ is a bond, O, S, NH or C(O). Preferably, the alkyl thio group is ofthe structure —S(CH₂)_(n)—, wherein n is an integer ranging from 2 toabout 4. More preferably, the thio group is of the structure—SCH₂CH₂C(O)—.

[0110] “Amino alkyl” refers to a chemical group having an amino groupattached to an alkyl group. Preferably an amino alkyl is of thestructure —(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10. More preferably it is of the structure —(CH₂)_(n)NH—, whereinn is an integer ranging from about 2 to about 4. Most preferably, theamino alkyl group is of the structure —(CH₂)₂NH—.

[0111] “Nucleic acid” refers to a polymer comprising 2 or morenucleotides and includes single-, double- and triple stranded polymers.“Nucleotide” refers to both naturally occurring and non-naturallyoccurring compounds and comprises a heterocyclic base, a sugar, and alinking group, preferably a phosphate ester. For example, structuralgroups may be added to the ribosyl or deoxyribosyl unit of thenucleotide, such as a methyl or allyl group at the 2′-O position or afluoro group that substitutes for the 2′-O group. The linking group,such as a phosphodiester, of the nucleic acid may be substituted ormodified, for example with methyl phosphonates or O-methyl phosphates.Bases and sugars can also be modified, as is known in the art. “Nucleicacid,” for the purposes of this disclosure, also includes “peptidenucleic acids” in which native or modified nucleic acid bases areattached to a polyamide backbone.

[0112] The phrase “coupled to a support” means bound directly orindirectly thereto including attachment by covalent binding, hydrogenbonding, ionic interaction, hydrophobic interaction, or otherwise.

[0113] “Probe” refers to a nucleic acid that can be used to detect, byhybridization, a target nucleic acid. Preferably, the probe iscomplementary to the target nucleic acid along the entire length of theprobe, but hybridization can occur in the presence of one or more basemismatches between probe and target.

[0114] “Perfect match probe” refers to a probe that has a sequence thatis perfectly complementary to a particular target sequence. The testprobe is typically perfectly complementary to a portion (subsequence) ofthe target sequence. The perfect match (PM) probe can be a “test probe”,a “normalization control” probe, an expression level control probe andthe like. A perfect match control or perfect match probe is, however,distinguished from a “mismatch control” or “mismatch probe.” In the caseof expression monitoring arrays, perfect match probes are typicallypreselected (designed) to be complementary to particular sequences orsubsequences of target nucleic acids (e.g., particular genes). Incontrast, in generic difference screening arrays, the particular targetsequences are typically unknown. In the latter case, prefect matchprobes cannot be preselected. The term perfect match probe in thiscontext is to distinguish that probe from a corresponding “mismatchcontrol” that differs from the perfect match in one or more particularpreselected nucleotides as described below.

[0115] “Mismatch control” or “mismatch probe”, in expression monitoringarrays, refers to probes whose sequence is deliberately selected not tobe perfectly complementary to a particular target sequence. For eachmismatch (MM) control in a high-density array there preferably exists acorresponding perfect match (PM) probe that is perfectly complementaryto the same particular target sequence. In “generic” (e.g., random,arbitrary, haphazard, etc.) arrays, since the target nucleic acid(s) areunknown perfect match and mismatch probes cannot be a priori determined,designed, or selected. In this instance, the probes are preferablyprovided as pairs where each pair of probes differ in one or morepreselected nucleotides. Thus, while it is not known a priori which ofthe probes in the pair is the perfect match, it is known that when oneprobe specifically hybridizes to a particular target sequence, the otherprobe of the pair will act as a mismatch control for that targetsequence. It will be appreciated that the perfect match and mismatchprobes need not be provided as pairs, but may be provided as largercollections (e.g., 3, 4, 5, or more) of probes that differ from eachother in particular preselected nucleotides. While the mismatch(s) maybe located anywhere in the mismatch probe, terminal mismatches are lessdesirable as a terminal mismatch is less likely to prevent hybridizationof the target sequence. In a particularly preferred embodiment, themismatch is located at or near the center of the probe such that themismatch is most likely to destabilize the duplex with the targetsequence under the test hybridization conditions. In a particularlypreferred embodiment, perfect matches differ from mismatch controls in asingle centrally-located nucleotide.

[0116] “Labeled moiety” refers to a moiety capable of being detected bythe various methods discussed herein or known in the art.

[0117] Nucleic Acid Labeling Compounds

[0118] The nucleic acid labeling compounds of the present invention areof the following structure:

A—O—CH₂—T—H_(c)—L—(M)_(m)—Q, or A—O—CH₂—T(H_(c))—L—(M)_(m)—Q, orA—O—CH₂—T—Q

[0119] wherein A is hydrogen or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; T isan optional template moiety; H_(c) is an heterocyclic group; L is alinker moiety; Q is a detectable moiety; and M is an connecting group,wherein m is an integer ranging from 0 to about 20. In a preferredembodiment m is from 0 to about 15. In a more preferred embodiment m isfrom 0 to about 10. In the most preferred embodiment m is from 0 toabout 5.

[0120] The group A is either hydrogen or a functional group that permitsthe attachment of a nucleic acid labeling compound to a nucleic acid.Nonlimiting examples of such groups include the following:monophosphate; diphosphate; triphosphate (H₄O₉P); phosphoramidite((R₂N)(R′O)P), wherein R is linear, branched or cyclic alkyl, and R′ isa protecting group such as 2-cyanoethyl; and H-phosphonate(HP(O)O—HNR₃), wherein R is linear, branched or cyclic alkyl.

[0121] The template moiety (T) is covalently attached to a methylenegroup (CH₂) at one position and a heterocyclic group (H_(c)) or thelinker moiety at another position. A nonlimiting set of templatemoieties is shown in FIG. 1, wherein the substituents are defined asfollows: X is O, S, NR₁ or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z isH, N₃, F or OR₁₀; W is O, S or CH₂; D is O or S; and, G is O, NH or CH₂.The substituents R₁, R₂, R₉ and R₁₀ are independent of one another andare H, alkyl or aryl.

[0122] The heterocyclic group (H_(c)) is a cyclic moiety containing bothcarbon and a heteroatom.

[0123] The linker moiety (L) of the nucleic acid labeling compound iscovalently bound to the heterocycle (H_(c)) or the template moiety atone terminal position. It is attached to the detectable moiety (O) atanother terminal position, either directly or through a connecting group(M). It is of a structure that is sterically and electronically suitablefor incorporation into a nucleic acid. Nonlimiting examples of linkermoieties include amido alkyl groups, functionalized alkyl groups,alkenyl alkyl groups, alkanoyl groups, and N-alkyl amido groups.

[0124] Amido groups have the structure —C(O)NR₃—, wherein R₃ ishydrogen, alkyl or aryl. Preferably, the amido group is of the structure—C(O)NR₃-where R3 is hydrogen or alkyl having from about 1 to about 6carbon atoms. More preferably, the amido alkyl group is of the structure—C(O)NH—.

[0125] Alkanoyl groups have the structure —(CH₂)_(n)C(O)—, n is aninteger ranging from 0 to about 10. Preferably, the alkanoyl group is ofthe structure —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 2 to about 10. More preferably, the alkanoyl group is of thestructure —(CH₂)_(n)C(O)—, wherein n is an integer ranging from about 2to about 6. Most preferably, the alkanoyl group is of the structure—CH₂C(O)—.

[0126] Amido alkyl groups have the structure —R₄C(O)NR₃—, wherein R₃ ishydrogen, alkyl or aryl, and R₄ is alkyl or aryl. The amido alkyl groupis preferably of the structure —(CH₂)_(n)C(O)NH—, wherein n is aninteger ranging from about 1 to about 10. More preferably, n is aninteger ranging from about 1 to about 6. Most preferably, the alkylamido group has the structure —(CH₂)₂C(O)NH— or the structure—CH₂C(O)NH—.

[0127] N-Amido alkyl groups have the structure —C(O)NR₃R₄—, wherein R₃is hydrogen, alkyl or aryl, and R₄ is alkylene or arylene. Preferably,the N-amido alkyl group is of the structure —C(O)NH(CH₂)_(n)R₅—, whereinn is an integer ranging from about 2 to about 10, and R₅ is O, NR₆, orC(O), and wherein R₆ is hydrogen, alkyl or aryl. More preferably, theN-amido alkyl group is of the structure —C(O)NH(CH₂)_(n)N(H)—, wherein nis an integer ranging from about 2 to about 6. Most preferably, theN-amido alkyl group is of the structure —C(O)NH(CH₂)₄N(H)—.

[0128] Alkenyl alkyl groups are of the structure —CH═CH—R₄—, wherein R₄is alkyl or aryl. The alkenyl alkyl group is preferably of the structure—CH═CH(CH₂)_(n)R₅—, wherein n is an integer ranging from 1 to about 10,and R₅ is O, NR₆, C(O) or C(O)NR₆, wherein R₆ is hydrogen, alkyl oraryl. More preferably, the alkenyl alkyl group is of the structure—CH═CH—(CH₂)_(n)C(O)NR₆—, wherein n is an integer ranging from 0 toabout 4. Most preferably, the alkenyl alkyl group is of the structure—CH═CH—C(O)N(H)—.

[0129] Functionalized alkyl groups are of the structure —(CH₂)_(n)R₇—,wherein n is an integer ranging from 1 to about 10, and R₇ is O, S, NH,or C(O). The functionalized alkyl group is preferably of the structure—(CH₂)_(n)C(O)—, wherein n is an integer ranging from 1 to about 4. Morepreferably, the functionalized alkyl group is —CH₂C(O)—.

[0130] Alkoxy groups are of the structure —O(CH₂)_(n)R₈—, wherein n isan integer ranging from 2 to about 10, and R₉ is O, S, NH, or C(O). Thealkoxy group is preferably of the structure —O(CH₂)_(n)— or—O(CH₂)_(n)C(O)—, wherein n is an integer ranging from 2 to about 4.More preferably, the alkoxy group is of the structure —OCH₂CH₂— or—OCH₂CH₂C(O)—.

[0131] Alkyl thio groups are of the structure —S(CH₂)_(n)R₈—, wherein nis an integer ranging from 2 to about 10, and R₈ is O, S, NH, or C(O).The alkyl thio group is preferably of the structure —S(CH₂)_(n)C(O)—,wherein n is an integer ranging from 2 to about 4. More preferably, thealkyl thio group is of the structure —SCH₂CH₂C(O)—.

[0132] Amino alkyl groups comprise an amino group attached to an alkylgroup. Preferably, amino alkyl groups are of the structure—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10. The amino alkyl group is more preferably of the structure—(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about 4.Most preferably, the amino alkyl group is of the structure —(CH₂)₄NH—.

[0133] The detectable moiety (O) is a chemical group that provides ansignal. The signal is detectable by any suitable means, includingspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. In certain cases, the signal is detectable by2 or more means.

[0134] The detectable moiety provides the signal either directly orindirectly. A direct signal is produced where the labeling groupspontaneously emits a signal, or generates a signal upon theintroduction of a suitable stimulus. Radiolabels, such as ³H, ¹²⁵I, ³⁵S,¹⁴C or ³²P, and magnetic particles, such as Dynabeads™, are nonlimitingexamples of groups that directly and spontaneously provide a signal.Labeling groups that directly provide a signal in the presence of astimulus include the following nonlimiting examples: colloidal gold(40-80 nm diameter), which scatters green light with high efficiency;fluorescent labels, such as fluorescein, texas red, rhodamine, and greenfluorescent protein (Molecular Probes, Eugene, Oreg.), which absorb andsubsequently emit light; chemiluminescent or bioluminescent labels, suchas luminol, lophine, acridine salts and luciferins, which areelectronically excited as the result of a chemical or biologicalreaction and subsequently emit light; spin labels, such as vanadium,copper, iron, manganese and nitroxide free radicals, which are detectedby electron spin resonance (ESR) spectroscopy; dyes, such as quinolinedyes, triarylmethane dyes and acridine dyes, which absorb specificwavelengths of light; and colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. See U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.

[0135] A detectable moiety provides an indirect signal where itinteracts with a second compound that spontaneously emits a signal, orgenerates a signal upon the introduction of a suitable stimulus. Biotin,for example, produces a signal by forming a conjugate with streptavidin,which is then detected. See Hybridization With Nucleic Acid Probes. InLaboratory Techniques in Biochemistry and Molecular Biology; Tijssen,P., Ed.; Elsevier: New York, 1993; Vol. 24. An enzyme, such ashorseradish peroxidase or alkaline phosphatase, that is attached to anantibody in a label-antibody-antibody as in an ELISA assay, alsoproduces an indirect signal.

[0136] A preferred detectable moiety is a fluorescent group. Flourescentgroups typically produce a high signal to noise ratio, thereby providingincreased resolution and sensitivity in a detection procedure.Preferably, the fluorescent group absorbs light with a wavelength aboveabout 300 nm, more preferably above about 350 nm, and most preferablyabove about 400 nm. The wavelength of the light emitted by thefluorescent group is preferably above about 310 nm, more preferablyabove about 360 nm, and most preferably above about 410 nm.

[0137] The fluorescent detectable moiety is selected from a variety ofstructural classes, including the following nonlimiting examples: 1- and2-aminonaphthalene, p,p′diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

[0138] A number of fluorescent compounds are suitable for incorporationinto the present invention. Nonlimiting examples of such compoundsinclude the following: dansyl chloride; fluoresceins, such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;N-phenyl-1-amino-8-sulfonatonaphthalene;N-phenyl-2-amino-6-sulfonatonaphthanlene;4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate; N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamin;N,N′-dioctadecyl oxacarbocycanine; N,N′-dihexyl oxacarbocyanine;merocyanine, 4-(3′-pyrenyl)butryate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene; 6-dimethylamino-1,2-benzophenzin; retinol;bis(3′-aminopyridinium)-1,10-decandiyl diiodide; sulfonaphthylhydrazoneof hellibrienin; chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-[p-(2-benzimidazolyl)phenyl]maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin; rosebengal and 2,4-diphenyl-3 (2H)-furanone. Preferably, the fluorescentdetectable moiety is a fluorescein or rhodamine dye.

[0139] Another preferred detectable moiety is colloidal gold. Thecolloidal gold particle is typically 40 to 80 nm in diameter. Thecolloidal gold may be attached to a labeling compound in a variety ofways. In one embodiment, the linker moiety of the nucleic acid labelingcompound terminates in a thiol group (—SH), and the thiol group isdirectly bound to colloidal gold through a dative bond. See Mirkin etal. Nature 1996, 382, 607-609. In another embodiment, it is attachedindirectly, for instance through the interaction between colloidal goldconjugates of antibiotin and a biotinylated labeling compound. Thedetection of the gold labeled compound may be enhanced through the useof a silver enhancement method. See Danscher et al. J. Histotech 1993,16, 201-207.

[0140] The connecting groups (M)_(m) may serve to covalently attach thelinker group (L) to the detectable moiety (O). Each M group can be thesame or different and can independently be any suitable structure thatwill not interfere with the function of the labeling compound.Nonlimiting examples of M groups include the following: amino alkyl,—CO(CH₂)₅NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH₂)_(i)O—,—(CH₂)_(j)NH—, —C(O)(CH₂)_(h)O—, —CO(CH₂)₅NHCO(CH₂)₅NH—,—NH(CH₂CH₂O)_(k)NH—, and —CO(CH₂)₅—; wherein, k is an integer from 1 toabout 5, preferably k is 1 or 2; m is an integer ranging from 0 to about5, preferably 0 to about 3; h, i and j are independently integers from 1to about 5, preferably 1 to about 3.

[0141] Synthesis of Nucleic Acid Labeling Compounds

[0142]FIG. 2 illustrates a synthetic route to nucleic acid labelingcompound, 4, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as the trimethyl silylethers according to the proceduredescribed in J. Antibiot. 1977, 30, 129. The N1 nitrogen atom isalkylated using a substituted iodoamide havng a biotin label attached,according to the procedure described in Tetrahedron 1984, 40, 33.(PEO-iodoacetyl biotin can be purchased from Pierce Chemical Co.) Thesilyl protecting groups are removed, providing a compound having freealcohol groups using acetic acid in methanol. The product is convertedinto a 5′-triphosphate to afford labeled compound 4.

[0143] FIGS. 3 illustrates a synthetic route to nucleic acid labelingcompounds, 6 and 7, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as the trimethyl silylethers according to the proceduredescribed in J. Antibiot. 1977, 30, 129. The N1 nitrogen atom isalkylated according to the procedure described in Tetrahedron 1984, 40,33, using ethyl iodoacetate. The ester is converted to amide, 5, usingethylene diamine. The biotin labeled compound is prepared by reactingamide, 5, with biotin-dPEG4™-NHS (purchased from Quanta Biodesign).Alternatively, amide 5 is reacted with 5-carboxyfluorescein-NHS. Thesilyl protecting groups are removed to provide a compound having freealcohol groups, using acetic acid in methanol. The product is convertedinto a 5′-triphosphate using standard phosphorylation conditions toafford, respectively, nucleic acid labeling compounds 6 and 7.

[0144]FIG. 4 illustrates an alternate synthetic route to nucleic acidlabeling compounds, 6 and 7, starting from2-amino-5-(β-D-ribofuranosyl)-4 (1H)-pyrimidinone, 1. The hydroxy groupsare protected as acetyl esters using acetic anhydride in pyridine. TheN1 nitrogen atom is alkylated according to the procedure described inTetrahedron 1984, 40, 33, using ethyl iodoacetate. The ester isconverted to amide, 8, using ethylene diamine. The biotin labeledcompound is prepared by reacting amide, 8, with biotin-dPEG4™-NHS(purchased from Quanta Biodesign). Alternatively, amide 8 is reactedwith 5-carboxyfluorescein-NHS. The silyl protecting groups are removedto provide a compound having free alcohol groups, using acetic acid inmethanol. The product is converted into a 5′-triphosphate using standardphosphorylation conditions to afford, respectively, nucleic acidlabeling compounds 6 and 7.

[0145]FIG. 5 illustrates a synthetic route to nucleic acid labelingcompounds, 10 and 11, starting from 2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groups are protected as acetyl estersusing acetic anhydride in pyridine. The N1 nitrogen atom is alkylatedaccording to the procedure described in K. Muhlegger, et al. 1996, WO96/28640, using methyl acrylate in triethyl amine. The ester, 8 isconverted to amide, 9, using ethylene diamine. The biotin labeledcompound is prepared by reacting amide, 9, with biotin-dPEG4™-NHS(purchased from Quanta Biodesign). Alternatively, amide 9 is reactedwith 5-carboxyfluorescein-NHS. The product is converted into a5′-triphosphate using standard phosphorylation conditions to afford,respectively, nucleic acid labeling compounds 10 and 11.

[0146]FIG. 6 illustrates a synthetic route to nucleic acid labelingcompounds, 14 and 15, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as acetyl esters using acetic anhydride in pyridine. TheN1 nitrogen atom is alkylated according to the procedure described inTet Lett 1995 36, 3261, using methyl propynoate. The ester, 12 isconverted to amide, 9, using ethylene diamine. The biotin labeledcompound is prepared by reacting amide, 9, with biotin-dPEG4™-NHS(purchased from Quanta Biodesign). Alternatively, amide 9 is reactedwith 5-carboxyfluorescein-NHS. The product is converted into a5′-triphosphate using standard phosphorylation conditions to afford,respectively, nucleic acid labeling compounds 14 and 15.

[0147]FIG. 7 illustrates a synthetic route to nucleic acid labelingcompounds, 17 and 18, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as the trimethyl silylethers according to the proceduredescribed in J. Antibiot. 1977, 30, 129. The N1 nitrogen atom isalkylated, using 2-(2-chloromethoxy-ethyl)-isoindole-1,3-dione,according to the procedure described in Chemo 1985, 31, 151. Theproduct, 16 is treated with hydrazine in ethanol to remove theprotecting groups. The biotin labeled compound is prepared by reactingthe ether-amine with biotin-dPEG4™-NHS (purchased from Quanta Biodesign)using standard conditions. Alternatively, the ether-amine is reactedwith (Ac)₂F1-NHS using standard conditions. The product is convertedinto a 5′-triphosphate using standard phosphorylation conditions toafford, respectively, nucleic acid labeling compounds 17 and 18.

[0148]FIG. 8 illustrates a synthetic route to nucleic acid labelingcompounds, 20 and 21, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as acetyl esters using acetic anhydride in pyridine. TheN1 nitrogen atom is alkylated using (6-trifluoroacetamido)hexyl alcoholin the presence of triphenyl phosphane ((Ph)₃P), anddiethylazodicarboxylate (DEAD), according to the procedure described inBrossette, T. et al., J. Org. Chem. 1999, 64, 5083 and Nuclesies,Nucleotides and Nucleic Acids 2000, 19, 867. The N1 nitrogen atom isalkylated using (6-trifluoroacetamido)hexyl alcohol according to theprocedure described in J. Antibiot. 1977, 30, 129. The protecting groupsare removed to provide a compound having free amine and alcohol groups,using ammonia in methanol. The biotin labeled compound is prepared byreacting the ether-amine with biotin-dPEG4™-NHS (purchased from QuantaBiodesign) using standard conditions. Alternatively, the ether-amine isreacted with (Ac)₂F1-NHS using standard conditions. The product isconverted into a 5′-triphosphate using standard phosphorylationconditions to afford, respectively, nucleic acid labeling compounds 20and 21.

[0149]FIG. 9 illustrates a synthetic route to nucleic acid labelingcompounds, 23 and 24, starting from2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 1. The hydroxy groupsare protected as acetyl esters using acetic anhydride in pyridine. TheN1 nitrogen atom is alkylated using 2-(6-iodo-hexyl)-isoindole-1,3-dioneaccording to the procedure described in J. Antibiot. 1977, 30, 129. Theproduct, 22 is treated with hydrazine in ethanol to remove theprotecting groups. The biotin labeled compound is prepared by reactingthe free amine with biotin-dPEG4™-NHS (purchased from Quanta Biodesign)using standard conditions. Alternatively, the ether-amine is reactedwith (Ac)₂F1-NHS using standard conditions. The product is convertedinto a 5′-triphosphate using standard phosphorylation conditions toafford, respectively, nucleic acid labeling compounds 23 and 24.

[0150]FIG. 10 illustrates a synthetic route to nucleic acid labelingcompounds, 36, starting from [1,4]Dioxan-2-one, Shahi, S. P., et al., J.Org. Chem. (1999), 64: 4509-11, Nishimura, T., et al., J. Org. Chem.1999, 64: 6750-55, Nishida, H., et al., J. Polym. Sci. 2000, 38:1560-67; (2-Hydroxy-ethoxy)-acetic acid methyl ester, Kitano, M.; andOhashi, N., EP 787728 A1(1997), or a polymer thereof, Nishida H, at al.J. Polym. Sci., (2000), 38: 1560-67, 33. The compound is reacted with adiamine having a suitable linker moiety, e.g., a polyethylene oxide,alkylene, a combination there of and the like. The labeled compound isprepared by reacting the ether-amine with a suitable labeling compoundusing standard conditions. The product is converted into a5′-triphosphate using standard phosphorylation conditions to afford,respectively, nucleic acid labeling compound 36.

[0151] Nucleic Acid Labeling

[0152] Nucleic acids can be isolated from a biological sample orsynthesized, on a solid support or in solution for example, according tomethods known to those of skill in the art. As used herein, there is nolimitation on the length or source of the nucleic acid used in alabeling process. Exemplary methods of nucleic acid isolation andpurification are described in Theory and Nucleic Acid Preparation. InLaboratory Techniques in Biochemistry and Molecular Biology.Hybridization With Nucleic Acid Probes; P. Tijssen, Ed.; Part I;Elsevier: N.Y., 1993. A preferred method of isolation involves an acidguanidinium-phenol-chloroform extraction followed by oligo dT columnchromatography or (dT)n magnetic bead use. Sambrook et al. MolecularCloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory,1989; Vols. 1-3; and Current Protocols in Molecular Biology; F. Ausubelet al. Eds.; Greene Publishing and Wiley Interscience: N.Y., 1987.

[0153] In certain cases, the nucleic acids are increased in quantitythrough amplification. Suitable amplification methods include, but arenot limited to, the following examples: polymerase chain reaction (PCR)(Innis, et al. PCR Protocols. A guide to Methods and Application;Academic Press: San Diego, 1990); ligase chain reaction (LCR) (Wu andWallace. Genomics 1989, 4, 560; Landgren, et al. Science 1988, 241,1077; and Barringer, et al. Gene 1990, 89, 117); transcriptionamplification (Kwoh et al. Proc. Natl. Acad. Sci. USA 1989, 86, 1173);and self-sustained sequence replication (Guatelli, et al. Proc. Nat.Acad. Sci. USA 1990, 87, 1874).

[0154] The nucleic acid labeling compound can be incorporated into anucleic acid using a number of methods. For example, it can be directlyattached to an original nucleic acid sample (e.g., MRNA, polyA mRNA,cDNA) or to an amplification product. Methods of attaching a labelingcompound to a nucleic acid include, without limitation, nicktranslation, 3-end-labeling, ligation, in vitro transcription (IVT) orrandom priming. Where the nucleic acid is an RNA, a labeledriboligonucleotide is ligated, for example, using an RNA ligase suchasT4 RNA Ligase. In The Enzymes; Uhlenbeck and Greensport, Eds.; Vol.XV, Part B, pp. 31-58; and, Sambrook et al., pp. 5.66-5.69. Terminaltransferase is used to add deoxy-, dideoxy- or ribonucleosidetriphosphates (dNTPs, ddNTPs or NTPs), for example, where the nucleicacid is single stranded DNA.

[0155] The labeling compound can also be incorporated at an internalposition of a nucleic acid. For example, PCR in the presence of alabeling compound provides an internally labeled amplification product.See, e.g., Yu et al. Nucleic Acids Research 1994, 22, 3226-3232.Similarly, IVT in the presence of a labeling compound can provide aninternally labeled nucleic acid.

[0156] Probe Hybridization

[0157] The nucleic acid to which the labeling compound is attached canbe detected after hybridization with a nucleic acid probe.Alternatively, the probe can be labeled, depending upon the experimentalscheme preferred by the user. The probe is a nucleic acid, or a modifiednucleic acid, that is either attached to a solid support or is insolution. It is complementary in structure to the labeled nucleic acidwith which it hybridizes. The solid support is of any suitable material,including polystyrene based beads and glass chips. In a preferredembodiment, the probe or target nucleic acid is attached to a glasschip, such as a GeneChip® product (Affymetrix, Inc., Santa Clara,Calif.). See International Publication Nos. WO 97/10365, WO 97/29212, WO97/27317, WO 95/11995, WO 90/15070, and U.S. Pat. Nos. 5,744,305 and5,445,934 which are hereby incorporated by reference.

[0158] Because probe hybridization is often a step in the detection of anucleic acid, the nucleic acid labeling compound must be of a structurethat does not substantially interfere with that process. The steric andelectronic nature of the labeling compound, therefore, is compatiblewith the binding of the attached nucleic acid to a complementarystructure.

EXAMPLES

[0159] The following examples are offered to illustrate, but not tolimit, the present invention.

[0160] General Experimental Details

[0161] Reagents were purchased from Aldrich Chemical Company (Milwaukee,Wis.) in the highest available purity. All listed solvents wereanhydrous. Intermediates were characterized by ¹H NMR and massspectrometry.

Example 1

[0162] Synthesis of fluorescein derivatives of 2′-amino-2′-deoxyuridinetriphosphate and 3′-amino-3′-deoxythymidinetriphosphate (Scheme 1)

[0163] To 0.5 umoles (50 uL of a 10 mM solution) of the amino nucleotidetriphosphate (1 or 2) in a 0.5 ml ependorf tube was added 25 ul of a 1 Maqueous solution of sodium borate, pH 8, 87 uL of methanol, and 88 uL(10 umol, 20 equiv) of a 100 mM solution of 5-carboxyfluorescein-X-NHSester in methanol. The mixture was vortexed briefly and allowed to standat room temperature in the dark for 15 hours. The sample was thenpurified by ion-exchange HPLC to afford the fluoresceinated derivatives3 or 4 in about 78-84% yield. Relative efficiencies of incorporation ofthese compounds by TdT are shown in Table 1.

TABLE 1 Incorporation of triphosphate compounds by TdT.

TdT Labeling Efficiencies % Labeled X(3') Y(2′) B(1′b) 40U 160U OH Huracil 100.0 100.0 NH2 H thymine 100 100 NHCO(CH2)5NH— H thymine 1.3 2.2(CO—FL) OH NH2 uracil 65 95 OH NHCO(CH2)5NH— uracil 3.0 6.6 (CO—FL) OHO(CH2)6NH—(CO—FL) uracil 2.5 7.0 OH O(CH2)6NHCO— uracil 15.0 17.0(CH2)5- NHCO-Biotin OH NH(CH2)5CH3 uracil 4.5 5.0

Example 2

[0164] Synthesis of N1-labeled2-Amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone

[0165] Synthesis of the N1-labeled 2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 55, involved alkylation at N1 using conditionssimilar to those described by Muehlegger, et al. (WO 96/28640) for theN1-alkylation of pyrazalo-[4,3-d]pyrimidines (FIG. 3A).

Example 3

[0166] Synthesis of N-(6-(fluorescein-5-carboxamido)hexanoyl)-morpholinouridine triphosphate (Scheme 2).

[0167] Morpholino-uracil tosylate salt 25 (30 mg) was co-evaporated withpyridine (3×3 ml) and dissolved in 2 ml of pyridine and cooled to 0° C.Trifluoroacetic anhydride (30 uL) was added and stirred for 1 hour. Thereaction was followed by HPLC until complete. The pyridine was removedand the residue was dissolved in 1 ml of water and filtered. The productwas purified by HPLC on a Waters C-18 bondapak cartridge (Buffer: A=50mM TEM pH 7.0; B=acetonitrile) using a gradient of 0-25%B in 30 minutes(retention time=22 min.). The product was desalted on a Sep-Pakcartridge and freeze-dried to give 151 mg of 26. Phosphorylation of 2using the POCl₃ method gave 27. The removal of the trifluoroacetyl groupwith conc. NH4OH at 50° C. for 30 min to provide the free amine, 28.Conjugation of 28 to 5-carboxyfluoroscein-aminocaproic acidN-hydroxysuccinimide (F1-X-NHS) under standard conditions provided amide29. The mass spectral and NMR data for compounds 25-29 were consistentwith the proposed structures.

Example 4

[0168] The compounds of the invention having general formulas I, IA IB,II, III, IIIA. IIIB, and IV as disclosed herein can be prepared usingavailable reagents and procedures that are standard in the art. Inaddition, examples of these procedures are illustrated in FIGS. 2-10.

Example 5

[0169] Procedure for HPLC Analysis of Enzymatic Incorporation ofModified Nucleotides.

[0170] Reaction Conditions

[0171] TdT

[0172] 3 uM dT₁₆ template

[0173] 15(30)uM NTP

[0174] 40 U TdT (Promega)

[0175] 1× buffer, pH 7.5 (Promega)

[0176] Procedure: incubate 1 hr. at 37° C., then for 10 min. at 70° C.,followed by the addition of EDTA (2 mM final concentration) in a volumeof 50 uL

[0177] HPLC Analysis

[0178] Materials and Reagents

[0179] 4.6 mm×250 mm Nucleopac PA-100 ion-exchange column (Dionex)buffer A: 20 mM NaOH (or 20 mM Tris pH 8, in the case of TdTincorporation of nucleotide triphoshates that are not dye-labeled)buffer B: 20 mM NaOH, 1M NaCl (or 20 mM Tris pH 8, 1M NaCl, in the caseof TdT incorporation of nucleotide triphoshates that are notdye-labeled)

[0180] General Procedure

[0181] Dilute the reaction with 50 uL of buffer A. Inject 50 uL of thissample onto the HPLC column and fractionate using a gradient of 5 to100% buffer B over 30 minutes at a flow rate of 1 mL/min. Detect thepeaks simultaneously at 260 nm absorbance and the absorbance maximum ofthe dye (or the fluorescence emission maximum of the dye).

[0182] The incorporation efficiency is expressed as the fraction ofoligonucleotide that is labeled. This number is determined by dividingthe peak area measured at 260 nm absorbance of the labeledoligonucleotide by the sum of the peak areas of the unlabeled andlabeled oligonucleotide. (The retention time of fluorescein-labeled dT₁₆is on the order of 2 to 3 min. longer than the unlabeled dT₁₆.) Theerror in this type of assay is about 10%. The percentage labelingefficiency for 4 types of nucleic acid labeling compounds is shown belowin Table 1.

Example 6

[0183] Labeled N-(2-hydroxyethoxy)ethyl 2-O-triphosphates (Scheme 3).

[0184] Compounds having general formula can be prepared using availablereagents and procedures that are standard in the art. In addition,examples of these procedures are illustrated in Schemes 3 and FIG. 10.

Example 6A

[0185] Labeled 2-(2-hydroxyethyl)acetamide 2-O-triphosphates (FIG. 10).

[0186] [1,4]Dioxan-2-one, (2-Hydroxy-ethoxy)-acetic acid methyl ester,or a polymer thereof, 33, is reacted with a diamine having a linkermoiety, e.g., a polyethylene oxide, alkylene, a combination there of.The labeled compound is prepared by reacting the ether-amine with asuitable labeling compound using standard conditions. The product isconverted into a 5′-triphosphate using standard phosphorylationconditions to afford, respectively, nucleic acid labeling compound 36.

Example 7

[0187] Synthesis of N-alkyl 2′-amino-2′-deoxyuridine triphosphate(Scheme 4).

[0188] 4,4-Diethoxy-butylamine is reacted with an activated N-labeledcaproyl amine, 37 to provide the N-labeled diethoxy butyl caproyl amide,38. The amide, 38, is reacted with aqueous hydrochloric acid (0.04 N) toproduce the N-labeled aldehyde. the aldehyde is reacted withphosphorylated1-(3-Amino-4-hydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2,4-dione,39, to furnish labeled N-alkyl 2′-amino-2′-deoxyuridine triphosphate,40. The reaction is illustrated in Scheme 4.

Example 8

[0189] Synthesis of 2′-O-(6-(Fluorescein-5-carboxamido)hexyl)uridine5′-O-triphosphate (Scheme 5).

[0190] The protected phthalimide, 41, (available from RI Chemicals, 500mg), is reacted successively with a) hydrazine/ethanol, b)Trifluoroacetic acid-imidazole at 0° C. and c) Acetic acid/methanol toprovide compound, 42. The alcohol-amine is converted into a5′-triphosphate using standard phosphorylation conditions followed byconjugation to a label to afford, respectively, nucleic acid labelingcompounds, 43.

Example 9

[0191] Synthesis of2′-S(N-(6-(Fluorescein-5-carboxamido)hexyl)aminoethyldithiouridine5′-O-triphosphate (Scheme 6).

[0192]3-Hydroxy-2-hydroxymethyl-2,3,3a,9a-tetrahydro-furo[2′,3′:4,5]oxazolo[3,2-a]pyrimidin-6-one,44, is treated with acetic anhydride in pyridine to protect fhe hydroxygroups. The protected compound, 45, is reacted with thioaceticacid/dioxane at 100° C., according to the procedure in J.Chem. Soc.Perkin Trans 1, 1997, 2587. the product is treated with ammonia providethiol, 46. The thiol is reacted with disulfide, 99, to providedisulfide, 47. the disulvide is converted into a 5′-triphosphate usingstandard phosphorylation conditions followed by reaction with a labeledreagent to afford, respectively, nucleic acid labeling compounds, 43.

[0193] All patents, patent applications, and literature cited in thespecification are hereby incorporated by reference in their entirety. Inthe case of any inconsistencies, the present disclosure, including anydefinitions therein will prevail.

[0194] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

1. A nucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, hydrogen, alkyl or aryl;Y is hydrogen, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is hydrogen, alkyl oraryl; Z is hydrogen, N₃, F, OR₉, SR₉ or NHR₁₀, wherein R₁₀ is hydrogen,alkyl or aryl; L is a linker moiety; Q is a detectable moiety; and M isa connecting group, wherein m is an integer ranging from 0 to about 20:2. The nucleic acid labeling compound of claim 1, wherein A is H orH₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is hydrogen, alkyl or aryl; Zis hydrogen, N₃, F or OR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl; andL is amido alkyl and M is —(M₁)_(a)—(M₂)_(b)— wherein a and b areindependently integers from 0 to about 5 and the sum of a and b is notgreater than
 15. 3. The nucleic acid labeling compound of claim 2,wherein L is —(CH₂)_(n)C(O)NR₃—, wherein R₃ is hydrogen, alkyl or aryland n is an integer ranging from about 1 to about 10; M₁ is —(CH₂)_(i)O—and M₂ is —(CH₂)_(j)NH—, wherein i and j are independently integers from1 to about
 5. 4. The nucleic acid labeling compound of claim 3, whereinY is H or OH; Z is H or OH; —L is —CH₂C(O)NH—; M₁ is —(CH₂CH₂O)₃— and M₂is —CH₂CH₂NH—; and Q is biotin or a carboxyfluorescein.
 5. The nucleicacid labeling compound of claim 4, wherein Y is OH; Z is OH; —L—(M)_(m)—is —CH₂C(O)NH—(CH₂CH₂O)₃—CH₂CH₂NH—; and Q is biotin.
 6. The nucleic acidlabeling compound of claim 4, wherein Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is carboxyfluorescein.
 7. The nucleicacid labeling compound of claim 1, wherein A is H or H₄O₉P₃—; X is O; Yis H or OR₉, wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, For OR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl; and L is amido alkyland M is —(M₁)_(a)—(M₂)_(b)—(M₃)_(c)—(M₄)_(d)— wherein a, b, c, and dare independently integers from 0 to about 5 and the sum of a, b, c, andd is not greater than
 15. 8. The nucleic acid labeling compound of claim7, wherein L is —(CH2)nC(O)NR3—, wherein R3 is hydrogen, alkyl or aryland n is an integer ranging from about 1 to about 10; each M isindependently —C(O)(CH2)kO—, —(CH2)iO— or —(CH2)jNH—, wherein i, j and kare independently integers from 1 to about
 5. 9. The nucleic acidlabeling compound of claim 8, wherein L is —CH₂C(O)NR₃—, wherein R₃ ishydrogen or, alkyl; M₁ is —(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃ is—(CH₂CH₂O)₃— and M₄ is (CH₂)₂NH—.
 10. The nucleic acid labeling compoundof claim 9, wherein Y is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—CH₂CH₂NH—C(O)CH₂CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin. 11.The nucleic acid labeling compound of claim 9, wherein Y is OH; Z is OH;—L—(M)_(m)— is —CH₂C(O)NH—CH₂CH₂NH—; m is 1; and Q iscarboxyfluorescein.
 12. The nucleic acid labeling compound of claim 8,wherein L is —(CH₂)₂C(O)NR₃—, wherein R₃ is hydrogen or, alkyl; M₁ is—(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃ is —(CH₂CH₂O)₃— and M₄ is—(CH₂)₂NH—.
 13. The nucleic acid labeling compound of claim 12, whereinY is OH; Z is OH; —L—(M)_(m)— is—CH₂C(O)NH—CH₂CH₂NH—C(O)CH₂CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin. 14.The nucleic acid labeling compound of claim 12, wherein Y is OH; Z isOH; —L—(M)_(m)— is —CH₂CH₂C(O)NH—CH₂CH₂NH—; m is 1; and Q iscarboxyfluorescein.
 15. The nucleic acid labeling compound of claim 1,wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ ishydrogen, alkyl or aryl; Z is hydrogen, N₃, F or OR₁₀ wherein R₁₀ ishydrogen, alkyl or aryl; and L is functionalized alkyl and M is—(M₁)_(a)—(M₂)_(b)—(M₃)_(c)—(M₂)_(d)— wherein a, b, c, and d areindependently integers from 0 to about 5 and the sum of a, b, c, and dis not greater than
 15. 16. The nucleic acid labeling compound of claim15, wherein L is —(CH₂)_(n)O—, wherein n is an integer ranging fromabout 1 to about 10; each M is independently —C(O)(CH₂)_(k)O—,—(CH₂)_(i)O— or —(CH₂)_(j)NH—, wherein i, j and k are independentlyintegers from 1 to about
 5. 17. The nucleic acid labeling compound ofclaim 16, wherein L is —CH₂O—; M₁ is —(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃is —(CH₂CH₂O)₃— and M₄ is —(CH₂)₂NH—.
 18. The nucleic acid labelingcompound of claim 17, wherein Y is OH; Z is OH; —L—(M)_(m)— is—CH₂O—CH₂CH₂NH—C(O)(CH₂)₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Q is biotin.
 19. Thenucleic acid labeling compound of claim 17, wherein Y is OH; Z is OH;—L—(M)_(m)— is —CH₂O—CH₂CH₂NH—; m is 1; and Q is carboxyfluorescein. 20.The nucleic acid labeling compound of claim 15, wherein L is—(CH₂)_(n)NR₃—, wherein R₃ is hydrogen, alkyl or aryl and n is aninteger ranging from about 1 to about 10; each M is independently—C(O)(CH₂)_(k)O—, —(CH₂)_(i)O— or —(CH₂)_(j)NH—, wherein i, j and k areindependently integers from 1 to about
 5. 21. The nucleic acid labelingcompound of claim 20, wherein L is —(CH₂)₆NH—; M₁ is —(CH₂)₂NH—, M₂ is—C(O)(CH₂)₂O—, and M₃ is —(CH₂CH₂O)₃—.
 22. The nucleic acid labelingcompound of claim 21, wherein Y is OH; Z is OH; —L—(M)_(m)— is—(CH₂)₆NH—C(O)(CH₂)₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; and Q is biotin.
 23. Thenucleic acid labeling compound of claim 21, wherein Y is OH; Z is OH; Lis —(CH₂)₆NH—; m is 0; and Q is carboxyfluorescein.
 24. The nucleic acidlabeling compound of claim 1, wherein A is H or H₄O₉P₃—; X is O; Y is Hor OR₉, wherein R₉ is hydrogen, alkyl or aryl; Z is hydrogen, N₃, F orOR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl; and L is alkenyl alkyl andM is —(M₁)_(a)—(M₂)_(b)—(M₃)_(c)—(M₄)_(d)— wherein a, b, c, and d areindependently integers from 0 to about 5 and the sum of a, b, c, and dis not greater than
 15. 25. The nucleic acid labeling compound of claim7, wherein L is—structure —CH═CH—(CH₂)_(n)C(O)N(R₆)—, wherein R₆ ishydrogen, alkyl or aryl and n is an integer ranging from about 0 toabout 10; each M is independently —C(O)(CH₂)_(k)O—, —(CH₂)_(i)O— or—(CH₂)_(j)NH—, wherein i, j and k are independently integers from 1 toabout
 5. 26. The nucleic acid labeling compound of claim 2, wherein L is—CH═CH—C(O)N(H); M₁ is —(CH₂)₂NH—, M₂ is —C(O)(CH₂)₂O—, M₃ is—(CH₂CH₂O)₃— and M₄ is —(CH₂)₂NH—.
 27. The nucleic acid labelingcompound of claim 12, wherein —L—(M)_(m)— is—CH═CH—C(O)NH—CH₂CH₂NH—C(O)CH₂CH₂O—(CH₂CH₂O)₃—CH₂CH₂NH—; Y is OH; Z isOH; Q is biotin.
 28. The nucleic acid labeling compound of claim 12,wherein Y is OH; Z is OH; —L—(M)_(m)— is —CH═CH—C(O)NH—CH₂CH₂NH—; m is1; and Q is carboxyfluorescein.
 29. The nucleic acid labeling compoundof claim 1, having the formula:

wherein A, X, Y, Z, L, M, m, and Q are as defined herein.
 30. A nucleicacid labeling compound of claim 1, having the formula:

wherein A, X, Y, Z, L, M, m, and Q are as defined herein.
 31. A nucleicacid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; L is a linkermoiety; Q is a detectable moiety; and M is a connecting group, wherein mis an integer ranging from 0 to about
 3. 32. The nucleic acid labelingcompound of claim 31, wherein A is H or H₄O₉P₃—; L is alkanoyl; and Q isbiotin or a carboxyfluorescein; M is —NR₃— where R₃ is hydrogen oralkyl, and wherein m is 1 or
 0. 33. The nucleic acid labeling compoundof claim 32, wherein L is —C(O)(CH₂)_(n)— wherein n is an integerranging from about 1 to about 10; M is —NH—; Q is biotin or acarboxyfluorescein.
 34. The nucleic acid labeling compound of claim 33,wherein —L—(M)_(m)— is —C(O)(CH₂)₆—NH—; and Q is biotin.
 35. The nucleicacid labeling compound of claim 33, wherein —L—(M)_(m)— is—C(O)(CH₂)₆—NH—; and Q is carboxyfluorescein.
 36. A nucleic acidlabeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X₁ is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, hydrogen, alkyl or aryl;R₁₅ is hydrogen, alkyl or aryl; Y₁ is hydrogen, N₃, F, OR₉, SR₉ or NHR₉,wherein R₉ is hydrogen, alkyl or aryl; Z₁ is hydrogen, N₃, F, OR₉, SR₉or NHR₁₀, wherein R₁₀ is hydrogen, alkyl or aryl; wherein one of Y, orZ, is a group having the formula —X₂L—(M), —Q; X₂ is O, S, or NR₁₆, andR₁₆ is hydrogen, alkyl or aryl; L is a linker moiety; Q is a detectablemoiety; and M is a connecting group, wherein m is an integer rangingfrom 0 to about
 20. 37. The nucleic acid labeling compound of claim 36,wherein A is H or H₄O₉P₃—; X₁ is O; Y₁ is hydrogen, OR₉, wherein R₉ ishydrogen, or alkyl, or —NR₁₆—L—(M)_(m)—Q; wherein R₁₆ is hydrogen, alkylor aryl; Z, is hydrogen OR₁₀, wherein R₁₀ is hydrogen, or alkyl, or—NR₁₆—L—(M)_(m)—Q, wherein R₁₆ is hydrogen, or alkyl; L is N-alkylamido; R₁₅ is hydrogen or alkyl; M is —(CH₂)_(n)NR₃-where R₃ is hydrogenor alkyl, and m is from 1 to about
 15. 38. The nucleic acid labelingcompound of claim 37, wherein Y, is hydrogen or OH; Z, is—NH—L—(M)_(m)—Q; L is —(CH₂)_(n)NHC(O)— where n is an integer from 1 toabout 10; M is —NH(CH₂)_(p)— where p is an integer from 1 to about 10;R₁₅ is hydrogen or methyl; and Q is biotin or a carboxyfluorescein. 39.The nucleic acid labeling compound of claim 38 wherein Y₁ is OH; R₁₅ ishydrogen; —L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin. 40.The nucleic acid labeling compound of claim 39, wherein Y₁ is OH; R₁₅ ishydrogen; —L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is acarboxyfluorescein.
 41. The nucleic acid labeling compound of claim 39wherein Y₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—;and Q is biotin.
 42. The nucleic acid labeling compound of claim 39,wherein Y₁ is OH; R₁₅ is methyl; —L—(M)_(m) is —(CH₂)₄NH—C(O)(CH₂)₅NH—;and Q is a carboxyfluorescein.
 43. The nucleic acid labeling compound ofclaim 37, wherein Y₁ is —NH—L—(M)_(m)—Q; Z₁ is hydrogen or OH; L is—(CH₂)_(n)NHC(O)— where n is an integer from 1 to about 10; M is—NH(CH₂)_(p)— where n is an integer from 1 to about 10; R₁₅ is hydrogenor methyl; and Q is biotin or a carboxyfluorescein.
 44. The nucleic acidlabeling compound of claim 43, wherein Z₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.
 45. The nucleicacid labeling compound of claim 43, wherein Z₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is a carboxyfluorescein.46. The nucleic acid labeling compound of claim 43, wherein Z₁ is OH;R₁₅ is methyl; —L—(M)_(m), is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is biotin.47. The nucleic acid labeling compound of claim 43, wherein Z₁ is OH;R₁₅ is methyl; —L—(M)_(m)— is —(CH₂)₄NH—C(O)(CH₂)₅NH—; and Q is acarboxyfluorescein.
 48. The nucleic acid labeling compound of claim 37,wherein A is H or H₄O₉P₃—; X₁ is O; Y₁ is hydrogen, OR₉, wherein R₉ ishydrogen, or alkyl, or —O—L—(M)_(m)—Q; Z, is hydrogen OR₁₀, wherein R₁₀is hydrogen, or alkyl, or —O—L—(M)_(m)—Q, or alkyl; L is alkylene; and mis from 1 to about
 10. 49. The nucleic acid labeling compound of claim48, wherein Y₁ is hydrogen or OH; Z₁ is —O—L—(M)_(m)—Q; L is —(CH₂)_(n)—where n is an integer from 1 to about 12; M is —NH—; R₁₅ is hydrogen ormethyl; and Q is biotin or a carboxyfluorescein.
 50. The nucleic acidlabeling compound of claim 49, wherein Y₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —(CH₂)₆—NH—; and Q is biotin.
 51. The nucleic acidlabeling compound of claim 49, wherein Y₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —(CH₂)₆—NH—; and Q is a carboxyfluorescein.
 52. Thenucleic acid labeling compound of claim 49, wherein Y₁ is OH; R₁₅ ismethyl; —L—(M)_(m)— is —(CH₂)₆—NH—; and Q is biotin.
 53. The nucleicacid labeling compound of claim 49, wherein Y₁ is OH; R₁₅ is methyl;—L—(M)_(m)— is —(CH₂)₆—NH—; and Q is a carboxyfluorescein.
 54. Thenucleic acid labeling compound of claim 37, wherein Y₁ is—O—L—(M)_(m)—Q; Z₁ is hydrogen or OH; L is —(CH₂)_(n)— where n is aninteger from 1 to about 12; M is —NH—; R₁₅ is hydrogen or methyl; and Qis biotin or a carboxyfluorescein.
 55. The nucleic acid labelingcompound of claim 54, wherein Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—(CH₂)₆—NH—; and Q is biotin.
 56. The nucleic acid labeling compound ofclaim 54, wherein Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is —(CH₂)₆—NH—;and Q is a carboxyfluorescein.
 57. The nucleic acid labeling compound ofclaim 54, wherein Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is —(CH₂)₆—NH—;and Q is biotin.
 58. The nucleic acid labeling compound of claim 54,wherein Z₁ is OH; R₁₅ is methyl; —L—(M)_(m)— is —(CH₂)₆—NHC—; and Q is acarboxyfluorescein.
 59. The nucleic acid labeling compound of claim 37,wherein A is H or H₄O₉P₃—; X₁ is O; Y₁ is hydrogen, OR₉, wherein R₉ ishydrogen, or alkyl, or —S—L—(M)_(m)—Q; Z₁ is hydrogen OR₁₀, wherein R₁₀is hydrogen, or alkyl, or —S—L—(M)_(m)—Q; L is alkyl thio; and m is from1 to about
 10. 60. The nucleic acid labeling compound of claim 59,wherein Y₁ is hydrogen or OH; Z₁ is —S—L—(M)_(m)—Q; L is —S—(CH₂)_(n)—where n is an integer from 1 to about 10; M is —NH—; R₁₅ is hydrogen ormethyl; and Q is biotin or a carboxyfluorescein.
 61. The nucleic acidlabeling compound of claim 60, wherein Y₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is biotin.
 62. The nucleic acidlabeling compound of claim 60, wherein Y₁ is OH; R₁₅ is hydrogen;—L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.
 63. Thenucleic acid labeling compound of claim 60, wherein Y₁ is OH; R₁₅ ismethyl; —L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is biotin.
 64. The nucleicacid labeling compound of claim 60, wherein Y₁ is OH; R₁₅ is methyl;—L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.
 65. Thenucleic acid labeling compound of claim 37, wherein Y₁ is—S—L—(M)_(m)—Q; Z₁ is hydrogen or OH; L is —S—(CH₂)_(n)— where n is aninteger from 1 to about 10; M is —NH—; R₁₅ is hydrogen or methyl; and Qis biotin or a carboxyfluorescein.
 66. The nucleic acid labelingcompound of claim 65, wherein Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m) is—S—(CH₂)₂—NH—; and Q is biotin.
 67. The nucleic acid labeling compoundof claim 65, wherein Z₁ is OH; R₁₅ is hydrogen; —L—(M)_(m)— is—S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.
 68. The nucleic acidlabeling compound of claim 65, wherein Z₁ is OH; R₁₅ is methyl;—L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is biotin.
 69. The nucleic acidlabeling compound of claim 65, wherein Z₁ is OH; R₁₅ is methyl;—L—(M)_(m)— is —S—(CH₂)₂—NH—; and Q is a carboxyfluorescein.
 70. Anucleic acid labeling compound having the formula:

wherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; X₁ is O, S, NR₁or CHR₂, wherein R₁ and R₂ are, independently, hydrogen, alkyl or aryl;X₂ is a bond or alkylene; Q is a detectable moiety; and G is aconnecting group.
 71. The nucleic acid labeling compound of claim 70,wherein A is H or H₄O₉P₃—; X₁ is O; X₂ is a bond; and G is—C(O)NR₃—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NR₃—, where R₃ is hydrogenor alkyl, and m and n are independently an integer from 1 to about 15.72. The nucleic acid labeling compound of claim 71, wherein G is—C(O)NH—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NH— where m is from 1 toabout 6 and n is from 1 to about
 4. 73. The nucleic acid labelingcompound of claim 70, wherein A is H or H₄O₉P₃—; X₁ is O; X₂ is CH₂; andG is —C(O)NR₃—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NR₃—, where R₃ ishydrogen or alkyl, and m and n are independently an integer from 1 toabout
 15. 74. The nucleic acid labeling compound of claim 73, wherein Gis —C(O)NH—(CH₂)_(m)—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)NH— wherein m is from 1 toabout 6 and n is from 1 to about
 4. 75. A nucleic acid derivativeproduced by coupling a nucleic acid labeling compound of claim 2, 7, 12,15, 20, 24, 32, 37, or 71 with a nucleic acid.
 76. A hybridizationproduct, wherein the hybridization product comprises the nucleic acidderivative of claim 75 bound to a complementary probe.
 77. Thehybridization product of claim 76, wherein the probe is attached to aglass chip.
 78. A hybridization product, wherein the hybridizationproduct comprises the nucleic acid derivative of claim 75 bound to acomplementary probe.
 79. A method of synthesizing a labeled nucleic acidcomprising attaching a nucleic acid labeling compound of claim 2, 7, 12,15, 20, 24, 32, 37, or 71 to a nucleic acid.
 80. A method of detecting anucleic acid comprising incubating a nucleic acid derivative accordingto claim 75 with a probe.
 81. A method according to claim 80, whereinthe probe is attached to a glass chip.